Abstract

Human immunodeficiency virus Nef is a myristoylated protein expressed early in infection by HIV. In addition to the well known down-regulation of the cell surface receptors CD4 and MHCI, Nef is able to alter T-cell signaling pathways. The ability to alter the cellular signaling pathways suggests that Nef can associate with signaling proteins. In the present report, we show that Nef can interact with calmodulin, the major intracellular receptor for calcium. Coimmunoprecipitation analyses with lysates from the NIH3T3 cell line constitutively expressing the native HIV-1 Nef protein revealed the presence of a stable Nef-calmodulin complex. When lysates from NIH3T3 cells were incubated with calmodulin-agarose beads in the presence of CaCl(2) or EGTA, calcium ion drastically enhanced the interaction between Nef and calmodulin, suggesting that the binding is under the influence of Ca(2+) signaling. Glutathione S-transferase-Nef fusion protein bound directly to calmodulin with high affinity. Using synthetic peptides based on the N-terminal sequence of Nef, we determined that within a 20-amino-acid N-terminal basic domain was sufficient for calmodulin binding. Furthermore, the myristoylated peptide bound to calmodulin with higher affinity than nonmyris-toylated form. Thus, the N-terminal myristoylation domain of Nef plays an important role in interacting with calmodulin. This domain is highly conserved in several HIV-1 Nef variants and resembles the N-terminal domain of NAP-22/CAP23, a myristoylated calmodulin-binder. These results for the interaction between HIV Nef and calmodulin in the cells suggested that the Nef might interfere with intracellular Ca(2+) signaling through calmodulin-mediated interactions in infected cells.

Immunoprecipitation analyses of Nef expressed in NIH3T3 cells. (A) Immunoblot analysis of Nef and calmodulin coimmunoprecipitated from lysates of NIH3T3 cells, which were permanently infected with Nef expression vector. Cell lysates were immunoprecipitated (IP) with the anti-Nef antibody (left lane) and a normal IgG used as a negative control (right lane), and were detected by anti-Nef (upper) and anti-CaM (lower) antibody, respectively. (B) NIH3T3 cells permanently expressing Nef were lysed and incubated with calmodulin-agarose beads (left) or agarose beads without calmodulin (right) in the presence of 2 mM CaCl2. After washing of the agarose beads, the bound proteins were resolved by SDS-PAGE and transferred to membrane. Blots were probed for anti-Nef antibody. (C) Cells were lysed, centrifuged to be separated into soluble and insoluble fraction, and incubated with calmodulin-agarose beads in the presence (left) or absence (right) of CaCl2. S and P represent the soluble fraction and the insoluble fraction, respectively. Blots were probed for anti-Nef antibody. Relative intensities of the bands are also shown under the gel image.

Direct interaction between GST-Nef and calmodulin. Aliquots containing GST (lanes 1,2) or GST fusion Nef proteins (lanes 3–6) were incubated with 50 μL of calmodulin-agarose beads in 20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 500 μM CaCl2 (lanes 1–4) or 4 mM EGTA (lanes 5,6) for 1 h at 25°C. After a short centrifugation, the supernatants were removed to analyze the unbound fractions (lanes 1,3,5). The protein-bound beads were then extensively washed with the above buffer and processed for SDS-PAGE as the bound fractions (lanes 2,4,6). Relative intensities of the bands are also shown under the gel image.

Analyses of binding of Nef to calmodulin using surface plasmon resonance. The calmodulin was trapped on the surface of a sensor chip containing covalently attached streptavidin. For analysis of calmodulin-Nef interaction, solutions of the Nef were injected across chip surfaces containing calmodulin. The running buffer contained 20 mM Tris-HCl, pH 7.4, 100 mM NaCl, 500 μM CaCl2 (A) or 25 mM EGTA (B). GST and GST-Nef were diluted in this buffer to a final concentration of 200 nM prior to injection. The volume of injected sample was 40 μL, and the flow rate was 10 μL/min.